44

DISCUSSION

The observations made during the present study, shall be discussed

under three headings for the convenience and better understanding.

1) The observations made on ethanol toxicity shall be

discussed and compared with the work of previous workers. If any

variations are observed during study, then possible explanation

shall be given.

2) The effects of different doses of green tea extract on

ethanol treated rats shall be discussed and compared with findings

of other workers. Similarities or variations observed during the

present study shall be tried to explain.

3) Effects of green tea extract shall be compared with the

normal control and ethanol treated control animals to drive at a

final conclusion.

Discussion on the Effects of Ethanol

Ethanol manifests its harmful effects either through direct

generation of reactive metabolites, including free radical species that

react with most of the cell components changing their structures and

functions, or by contributing to other mechanisms that finally promote

enhance oxidative damage (Kato et al., 1990; Nordmann, 1994). The liver

is the major target organ of ethanol toxicity, and the role of oxidative

stress in the pathogenesis of alcohol-related disease, particularly in the

liver, has been repeatedly confirmed (Lieber, 1997).

45

Discussion on the effects of ethanol on body weight and organ weight

During the present study ethanol treatment caused decrease in the

body weight with 0.25 ml and 0.5 ml doses. The loss in body weight was

not significant even after 30 days treatment. There was a non significant

decrease in the mean body weight of rats treated with ethanol as

compared with the control group. Comparison between the initial and

final weights of rats in each group showed that rats in the control group

had a mean increase of 8.61 % in body weight while rats treated with 0.25

ml per 100g body weight of ethanol (50% v/v) showed body weight mean

-0.70 %.

Feinman (1998) reported that excessive alcohol intake can impair

the utilization of nutrients by altering their storage and excretion. Alcohol

impairs nutrient absorption by damaging cells lining the stomach and

intestine, and disabling transport of some nutrients into the blood. The

decrease in body weight can be attributed to the effects of alcohol on

digestion, absorption, storage, utilization and excretion of essential

nutrients such as vitamins, minerals and proteins (Lieber, 2003). Alcohol

also inhibits the breakdown of nutrients into usable substances, by

decreasing the secretion of digestive enzymes from the pancreas

(Korsten, 1989). Alcohol also impairs the utilization of nutrients by

altering their storage and excretion. Venukumar and Latha (2004) have

reported loss in body weight in carbon tetra chloride treated rats. The

metabolic dysfunction due to hepatopathy was believed to be the

causative factor for the significant decline in percentage weight gain in

animals with administration of carbon tetrachloride. Nagaraja et al.

(2006) also reported decrease in the body weight of alcohol treated rats.

However, Geidam (2007) have reported slight increase in the body

46

weight although this gain was statistically insignificant. Dahiru and

Obidoa (2007) observed significant decrease in body weight of rats

treated with alcohol for four weeks. Other researchers earlier reported

significantly decreased body weight due to alcohol comparison as

compared to control rats (Aruna et al., 2005, Das et al., 2006).

Osonuga et al. (2010) have also reported no significant change in

body weights of ethanol treated rats when compared with the controls.

This trend was also observed in the recovery group. The non significant

change in body weights of the ethanol treated rats may be because of the

short duration of ethanol administration. Our findings are similar to these

findings. Arulmozhi et al. (2010) also found that body weight was

significantly reduced in the ethanol-treated rats as compared to the

controls. Macdonald et al. (2010) have also reported no significant

decrease in mean body weight of rats treated with ethanol.

It can be concluded that decrease in mean body weight of alcohol

control group is mainly due to the fact that alcohol consumption results in

malnutrition due to reduced absorption of nutrients from the intestine.

On the other hand an increase in liver weight was observed both in

0.25 ml and 0.5 ml dose groups. The increase in liver weight was more

after 30 days of alcohol consumption.The increase in the weight of liver

is attributed to the fact that chronic alcohol consumption causes

accumulation of lipids and proteins in hepatocytes. The liver can be

injured by many chemicals and drugs. In the present study ethanol was

selected as a hepatotoxicant to induce liver damage, since it is clinically

relevant.

47

Ethanol produces a constellation of dose-related deleterious effects

in the liver (Leo et al., 1982). In chronic alcoholics, hepatomegaly occurs

due to the accumulation of lipids and proteins in the hepatocytes,

(Ashakumary et. al., 1993) with an impaired protein secretion by

hepatocytes (Vilstrup et al., 1985). Water is retained in the cytoplasm of

hepatocytes leading to enlargement of liver cells resulting in increased

total liver mass and volume (Blancho et al., 1990). Gujarati et al. (2006)

reported increase in the weight of liver of rats treated with 3.76 g/kg

ethanol for 25 days. Significant increase in the weight of liver was

reported with intraperitoneal injection with 1g/Kg body weight and 2g/Kg

body weight ethanol by Nagaraja et al. (2006). Javed et al. (2008)

observed increase in the liver weight of quails with 2% ethanol but

decrease in the liver weight after 4 weeks. After statistical analysis they

have concluded no significant difference from control groups. Arulmozhi

et al. (2010) also found that the liver weight was increased in animals fed

with ethanol as compared to the controls. Naik et al. (2011) reported that

carbon tetrachloride treatment significantly increased the relative weight

of liver of rats, which may be due to hepatomegaly or hepatotrophy.

Chronic ethanol administration is known to enhance the protein and fat

accumulation in the liver. Ethanol and acetaldehyde are known to induce

cell injury and necrosis by enhancing the production of reactive oxygen

species by the process of lipid peroxidation (Kurose et al., 1996).

Discussion on the effects of ethanol on liver biochemistry

As liver is the chief site of metabolism, liver is the first organ

affected with toxicity of any pollutant and toxicant hence it was thought

worthwhile to include the liver for biochemical analysis. So liver is the

main target organ of activity of any toxicant or drug or pollutant. During

48

the present study level of proteins, cholesterol and enzymes like

glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate

transaminase (GOT) were evaluated in liver.

In the ethanol control groups, significant decrease in level of the

total proteins in liver was observed with 0.25 ml and 0.5 ml doses of

ethanol of all durations.

Decreased level of serum protein and albumin is indicative of liver

damage. This damage is attributed to the fact that higher concentration of

alcohol dehydrogenase enzyme which catalyses alcohol to aldehyde

(Tussy and felder, 1989). Chronic alcohol use results in the accumulation

of export type proteins due to inhibition of the secretion of the proteins

from liver. It results in decreased level of proteins (Baraona, 1982).

Ethanol induced injury at least in part is attributed to an oxidative stress

resulting from the increase in free radical production and /or decrease

antioxidant defense. Dubey et al. (1994) reported that capacity of liver to

synthesize albumin is adversely affected by hepatotoxins. They reported

low level of albumin in alcoholic cirrhosis. The lowered level of total

proteins in the liver of ethanol treated rats can be attributed to this fact

only. It is also worthwhile to mention here that level of albumin is highly

significantly reduced in liver whereas globulin as not much affected.

Inhibition of protein synthesis is an important indication of cellular

damage. Decreased level of proteins also indicates that new cells are not

being formed. Arun Raj et al. (2009) have also observed decreased level

of serum albumin. Lower level of serum albumin may be due to reduced

liver function.

49

During the present project significant increase was observed in

liver cholesterol level in the liver tissue homogenate, after 15 and 30 days

treatment with both 0.25 ml and 0.5 ml doses to different groups of rats.

Ethanol is a powerful indicator of hyperlipidemia in both animals

and humans (Avogaro P, et al., 1975). The most common lipid

abnormalities during chronic alcohol consumption are known to produce

hypercholesterolemia and hypertriglyceridemia (Baraona et al., 1979;

1983).

Nagaraja et al. (2006) observed significant decrease in cholesterol

level after 7 (P<0.001) and 14 days (P<0.001) of ethanol administration

in unstressed animals. Serum cholesterol level significantly decreased

more after 2g/Kg body weight ethanol treatment compared to 1g/Kg body

weight ethanol (P<0.01). But our results are not in agreement with these

results. Arun Raj et al.(2009) have also observed increased cholesterol in

the liver, which is attributed to increased hepatic synthesis of cholesterol

induced by ethanol consumption (Leiber C.S. and Davidson C.S., 1962).

Ethanol induces hypercholesteremia which may be due to the activation

of enzyme HMG CoA reductase, the rate limiting step in cholesterol

biosynthesis (Younes et al., 1987). Arulmozhi et al. (2010) studied the

level of total cholesterol in ethanol-fed rats and found significant

increase. Our results are in agreement of these results.

During the present project significant increase was observed in

ALT and AST levels in the liver tissue homogenate, after 15 and 30 days

treatment with 0.25 ml and 0.5 ml doses of ethyl alcohol.

Soliman et al. (2006) reported decreased level of ALT and AST.

They have explained the decreased level of these enzymes due to toxic

50

action of ethanol on proteins and hepatocyte cellular organelles in general

but our studies are not in agreement with this work.

Dahiru et al. (2007) reported that exposure of rats to alcohol for six

weeks caused elevation of ALT and AST. Our results are in agreement of

these results.

The present study indicates that chronic ethanol treatment alters

biochemical parameters, however, the degrees of changes depend on the

doses and duration of ethanol treatment.

Discussion on the effects of ethanol on various blood

parameters

Since erythrocytes circulating in the peripheral blood share in part

structural and functional features common to most cells, it was thought

worthwhile to study the effect of ethanol on blood cells. During the

present study haemoglobin percentage (Hb%), red blood cells count

(RBCs count), packed cell volume percentage (PCV%), mean corpuscular

volume (MCV), mean corpuscular haemoglobin (MCH), mean

corpuscular haemoglobin concentration (MCHC), total leucocyte count

(TLC), differential leucocyte count (DLC), serum glucose, serum

proteins, serum albumin, serum globulin, blood urea, serum cholesterol,

serum bilirubin, acid phosphatase (ACP), alkaline phosphatase (ALP),

serum alanine transaminase (ALT) or serum glutamate pyruvate

transaminase (SGPT) and serum aspartate transaminase (AST) or serum

glutamate oxaloacetate transaminase (SGOT) were evaluated in the

blood of albino rats treated with 0.25 ml and 0.5 ml/100 gm body weight

doses of the ethanol for different durations.

51

During the present project, there was significant decrease (p< 0.05)

observed in haemoglobin percentage after the administration of both 0.25

ml and 0.5 ml doses of ethanol for 15 days and 30 days treatment.

Erythrocytes showed significant (p<0.05) decrease in number after 15 and

30 days treatment with 0.25 ml and 0.5 ml doses.

The reduced number of RBCs and decreased haemoglobin

percentage are most probably due to suppressive effect of ethanol on

erythrogenic tissues or destruction of RBCs by altering membrane

function of erythrocytes. Halliwell B. and Gutteridge J.M., (1986)

reported ethanol-induced liver injury may be linked, at least in part, to an

oxidative stress resulting from increased free radical production and/or

decreased antioxidant defence, though erythrocytes are prone to oxidative

damage due to presence of polyunsaturated fatty acids (PUFA), heme,

iron and oxygen. Parmahamsa, Rameswara Reddy, & Varadacharyulu,

(2004); Reddy et al. (2009) reported copious ethanol intake was

associated with the release of nitric oxide and induced the generation of

peroxynitrite and free radicals. Red blood cells were found to be very

sensitive to the above mentioned metabolites which induced oxidation of

membrane lipids, proteins and enhances fragility. Reddy et al. (2009)

reported that chronic ethanol intake causes many cellular alterations,

particularly in erythrocytes. Latvala et al. (2001); Tyulina et al. (2006)

also reported that ethanol metabolism which occurred, in major part, in

liver led to the formation of acetaldehyde, a highly cytotoxic compound

responsible for the oxidation of proteins, erythrocyte abnormalities and

hemolysis.

The destructive or suppressive effects of alcohol on erythrogenic

tissue can be expressed as a failure in red cell production and in

52

accelerated destruction of peripheral erythrocytes in the presence of an

abnormal cell population. Ethanol metabolism in the liver leads to the

formation of acetaldehyde which is highly cytotoxic compound and is

responsible for haemolyis and abnormalities in erythrocytes. Alcohol

intake is associated with the release of nitric oxide which causes the

generation of free radicals. Free radicals cause oxidation of membrane

lipids, proteins and enhanced fragility of RBCs (Tyulina 2006).

Osonuga et al. (2010) have reported significant decrease in the

level of Hb% and decreased RBCs count with 0.6 ml dose of ethanol. So

ethanol induces anemia due to destruction of Hb% and RBCs. Hence

people prone to anemic tendencies should abstain from any form of

alcohol. Mongi Saoudi et al. (2011) observed that the methanol treatment

induce significant decrease in the red blood cell (RBCs) and haemoglobin

(Hb%). Our results are in agreement with these results.

PCV levels also showed significant decrease in both groups (p<

0.05) and all durations with both 0.25 ml and 0.5 ml doses of ethanol

doses of ethanol.

Osonuga et al. (2010) have reported significant decrease in the

level of PCV% with 0.6 ml dose of ethanol. Our results are consistent

with their work.

MCV and MCH showed significant (p<0.05) decrease in both sets

after 15 and 30 days treatment with 0.25 ml dose and 0.5 ml doses

whereas MCHC showed no significant decrease in both sets. As MCV,

MCH and MCHC are calculated from haemoglobin percentage, PCV and

RBC count, so it is natural that these values will change accordingly.

53

A significant (P<0.05) decrease was also observed in TLC with

ethanol treatment for 15 and 30 days after the administration of 0.25 ml

and 0.5 ml doses of ethanol.

Our results are consistent with Osonuga et al. (2010), who studied

the effects of ethanol on haematological parameters in albino rats. The

rats were treated with 0.6 ml/200 gm/ body weight of the 25% ethanol for

3 days through oral rout of administration and observed the significant

reduction in TLC.

As TLC was decreased, so naturally DLC was also decreased but

all cell types are not equally affected. Ethanol treatment in rats

significantly decreased neutrophil and eosinophil counts in both sets and

durations of treatment. There was a significant decrease in absolute

neutrophil and eosinophil counts after chronic ethanol administration with

0.25 ml dose for a treatment period of 15 and 30 days. Administration of

0.5 ml dose of ethanol resulted in more significant decrease in absolute

neutrophil (p<0.05) and absolute eosinophil counts (p<0.01). No

significant change was observed in the number of basophils, monocytes

and lymphocytes.

Eichner and Hillman (1971) observed that ethanol depresses the

hemopoiesis in an organism by producing vacuolation in the granulocyte

precursors in the bone marrow. Neutropenia has been reported in patients

consuming alcohol. Sipp et al. (1993) have reported that eosinopenia

after ethanol treatment may be due to the direct effect of ethanol on

adrenal gland secretion. Ethanol-induced corticosterone secretion might

be the cause of decreased eosinophil count. Nagaraja et al. (2006)

observed a significant decrease (p<0.001) in absolute neutrophil and

eosinophil counts after chronic ethanol administration for a treatment

54

period of 7 and 14 days in unstressed rats when compared to control

groups . Administration of ethanol at a dosage of 2g/kg body weight

resulted in more significant decrease in absolute neutrophil (p<0.05) and

absolute eosinophil counts (p<0.01 -7 days; p<0.05 -14 days). Our

findings are also in accordance with the findings of these researchers.

The level of blood sugar showed significant increase (p<0.05) after

15 and 30 days treatment with 0.25 ml and 0.5 ml doses.

Arun Raj et al. (2009) studied the biochemical effects of feeding

soft drink and ethanol. Rats were fed intragastrically 3.0 ml/100 g body

weight rum diluted with water equivalent to 0.5 g ethanol and observed

significantly higher levels of glucose (p<0.05) compared to control rats.

Mongi Saoudi et al. (2011) have also reported increased glucose level

with methanol toxicity. Our findings are in accordance with these

research results.

During the present study serum protein and albumin levels were

decreased significantly (p<0.05) due to ethanol exposure for 15 and 30

days with both doses, but showed no effect on globulin level. Baraona E.

and Lieber C.S., (1982) reported ethanol inhibits the secretion of proteins

from the liver. Chronic ethanol abuse resulted in intrahepatic

accumulation of export-type proteins which results in decreased plasma

levels. These effects appear to be mediated by acetaldehyde. Das S.K.

and Vasudevan D.M., (2005) reported decreased serum protein and

albumin levels and indicated that liver damage starts after 30 days of

ethanol exposure. The common feature of chronic alcoholic liver disease

is progressive hypoalbuminemia.

55

Geidam et al. (2007) observed significantly decreased total protein

and albumin levels due to ethanol exposure in comparision to rats of

control group. Das et al. (2009), Gujarati et al. (2006) have also observed

that serum protein and albumin levels were decreased significantly

(p<0.001) due to ethanol exposure when compared with rats of control

group, but showed no effect on globulin levels. Arun Raj et al.(2009)

have also observed decreased level of serum albumin. Lower level of

serum albumin may be due to reduced liver function. Mongi Saoudi et al.

(2011) also studied the methanol-induced significant decrease in the

levels of serum total protein. Our results are in agreement with the

findings of all above research groups.

The level of urea showed no change after 15 and 30 days treatment

with both doses. Our results are consistent with Das et al. (2009) who

also observed no changes in the blood urea level till 4 weeks and present

work duration was also of 30 days.

During the present study significant increase (p<0.001) was

observed in the level of serum cholesterol after 30 days treatment with

0.25 and 0.5 ml doses/100 gm body weight of rats.

Leiber C.S. and Davideon C.S., (1962) studied the metabolic

effects of ethyl alcohol and according to them ethanol is known to

increase hepatic synthesis of cholesterol. The increased cholesterol during

alcohol ingestion is attributed to the increased β-hydroxyl methyl glutaryl

CoA (HMG CoA) reductase activity, which is the rate limiting step in

cholesterol biosynthesis (Ashakumari L. and Vijyammal P.L., 1993). Our

findings are in agreement with the findings of Gujrati et al. (2006), who

observed that ethanol administration to rats resulted in significant

elevation of total cholesterol in blood in comparison to control group.

56

Arun Raj et al.(2009) and Arulmozhi et al. (2010), have also observed

significant elevation in the total cholesterol with different doses of

alcohol. Mongi Saoudi et al. (2011) studied the methanol-induced

haematological and biochemical perturbation and observed significantly

increased cholesterol level in serum of treated animals.

The level of bilirubin showed significant increase (p<0.01) after 15

and 30 days treatment with 0.25 ml and 0.5 ml doses of ethyl alcohol.

Gujrati et al. (2006) and Dahiru and Obidoa (2007) observed that ethanol

administration to rats resulted in significant elevation of total serum

bilirubin when compared with control group. Our results are also

consistent with Maruthi et al., 2010 and Wan-Guo Yu et al. 2011.

During the present study level of acid phosphatase in blood showed

increase after 15 and 30 days treatment in 0.25 ml and 0.5 ml dose groups

of rats. Acid phosphatase is a lysosomal enzyme which hydrolyses the

ester linkage in phosphate esters and helps in the autolysis of the cell after

death. Experimental evidences show that it is not only restricted to

lysosomal fraction but has also been found in golgi cisternae and

specialized region of endoplasmic reticulum. Acid phosphatase reaction

thus reflects some impressions about the structure and function of these

organelles or components. Ress and Sinha (1960) are of the opinion that

the damaged organs might produce an augmented quantity of the

enzymes. Soliman et al. (2006) observed that ethanol ingestion caused

significant elevation in the activity of enzyme acid phosphatase. Geidam

et al. (2007) found mild elevation in acid phosphatase enzyme in ethanol

fed rats.

During the present study level of activities of liver specific

enzymes such as ALP, ALT and AST were increased significantly in

57

response to 15 and 30 days duration of ethanol exposure with both doses.

ALP, ALT and AST are the marker enzymes of liver damage. Increased

level of these enzymes in blood indicates liver damage. Gujrati et al.

(2006) reported that ethanol administration significantly elevates the

levels of ALP, ALT and AST enzymes in blood. Dahiru et al. (2007)

observed that the rats receiving alcohol only showed significantly

(p<0.05) elevated levels of ALT and AST as compared to control rats.

Arun Raj et al. (2009) observed the higher level of ALP, ALT and AST

after ethanol treatment in rats in comparison to control group of rats. The

level of significance (p<0.01) of these enzymes was same. Das et al.

(2009) studied the significant increase in the level of ALP, AST and ALT

in response to duration of ethanol exposure.

Elevated activities of these enzymes indicate liver damage. This is

because of higher concentration of alcohol dehydrogenase in liver, which

catalyzes alcohol to its corresponding aldehyde (Tussey et al. 1989). Our

results are also consistent with Halliwell B. and Gutteridge J.M., (1986);

Maruthi et al., (2010); Jingyu Yang et al. (2010) and Wan-Guo Yu et al.

(2011) who have also observed the higher level of ALP, ALT and AST

enzymes after CCl4 treatment in rats in comparison to control group of

rats.

Assessment of liver function can be made by estimating the

activities of serum ALP, AST and ALT which are enzymes originally

present in higher concentration in the cytoplasm. When there is damage

to liver or any other organ, these enzymes leak into the blood stream in

conformity with the extent of liver damage or damage to any other organ.

ALT and AST are most extensively studied enzymes for liver function.

58

Higher is the level of these enzymes in the blood more is the extent of

liver damage.

Discussion on the Effects of Different Doses of

Aqueous Extract of C. sinensis Co-treated with

Different Doses of Ethanol

Many polyphenols and flavanols are found in Camellia sinensis.

Several studies have proved that these polyphenols (catechins) and

flavanols have strong antioxidant activity and possess protective effect on

human health. It has also been reported that flavanols have potent

antioxidant properties including the scavenging of oxygen radicals and

lipid radicals. Several animal models have been used to test antioxidant

activity of flavonols from time to time but none has been tested for its

protective effect against ethanol toxicity.

During the present study aqueous extract of C. sinensis was used in

albino rats in two doses i.e. 5 mg/100 g body weight and 10 mg/100 g.

body weight doses. For studying protective effect of C. sinensis against

ethanol toxicity, experimental animals were treated with C. sinensis

aqueous extract along with 0.25 ml and 0.5 ml doses of ethanol in two

sets. For studying whether the effect of C. sinensis is temporary or

permanent, extract feeding was stopped and rats were given normal diet

for 15 more days.

During the present project, we studied several haematological

parameters and the biochemistry of liver. Blood was analyzed for Hb %,

RBC count, PCV%, MCV, MCH, MCHC%, TLC, DLC, sugar, total

proteins, albumin, globulin, urea, cholesterol, bilirubin, alkaline

phosphatase, acid phosphatase, serum ALT and serum AST.

59

While discussing the results, it is customary to compare the

findings with parallel work of other researchers. As we were not able to

find any work on ethyl alcohol + C. sinensis aqueous extract, we have

compared our results with effects of C. sinensis with other chemicals or

effects of ethyl alcohol with other antioxidant plants, where ever possible.

Discussion on the effects on body weight and organ weight

During the present study increase in body weight was observed

after 15 and 30 days treatment with 5 mg and 10 mg doses of aqueous

extract of C. sinensis. In all treatment groups body weight showed

increase although percentage increase was different in different treatment

groups and durations. When we compare these readings with those of the

normal control groups of the same duration, almost similar weight gain

pattern was observed.

But if we compare the body weight by ethanol and green tea, then

it is clear that body weight was different after 15 and 30 days treatment.

The body weight with 0.25 ml ethanol was 0.70% after 15 days treatment

and 4.70% after 30 days treatment and whereas with 5 mg C. sinensis

dose the body weight was 6.24% and 7.15% and with 10 mg dose it was

8.34% and 8.46% after 15 days and 30 days treatment respectively. On

the other hand in 0.5 ml ethanol group, loss of body weight was 4.12%

after 15 days treatment and 2.93% after 30 days treatment. In 5 mg C.

sinensis group body weight was 5.71% and 8.38% after 15 and 30 days

respectively. In 10 mg dose treatment the body weight was 7.33% and

8.98% after 15 and 30 days respectively.

These percentages of weight loss indicate that chronic treatment

with ethanol causes loss in body weight and percentage loss increases

60

with increase in dose and duration of the ethanol treatment. On the other

hand percentage of body weight increase with C. sinensis doses was

different with different doses and durations.

During the present project, reversibility studies were also

conducted to see whether the effect of C. sinensis is temporary or

permanent. In set I, group 2 (ii), when ethanol treatment was discontinued

for 15 days, the loss in body weight was 4.71% and in set I, group 2 (iv),

when ethanol treatment was discontinued for 15 days after 30 days

ethanol feeding, the loss in body weight was 5.61%. When we compare

these readings with C. sinensis fed groups, in set I, group 3 (ii), the body

weight was 6.08%and in set I, group 3 (iv), the body weight was 6.08%.

In set I, group 4 (ii), the body weight was 7.60% and in set I, group 4 (iv),

the body weight was 8.46% only.

In set II, group 2 (ii), where 0.5 ml ethanol was given for 15 days

and then discontinued for 15 days, the body weight was 4.24%. In set II,

group 3 (ii), the body weight was 6.42%. In set II, group 4 (ii) where 10

mg C. sinensis was given for 15 days and discontinued for 15 days, the

body weight was 8.80%. In 30 days reversibility group of 0.5 ml ethanol

+15 days normal diet, the body weight was 4.28%. In set II, group 3 (iv),

the body weight was 7.17% and in set II, group 4 (iv), the body weight

was 8.98%.

The body weights of ethanol treated rats showed decrease. There

was a decrease in the mean body weight of rats treated with ethanol as

compared with the control group. Comparison between the initial and

final weights of rats in each group showed that rats in the control group

had a mean % increase of 8.61 % in body weight while rats treated with

0.25ml per 100g body weight of ethanol (50% v/v) showed only 0.70 %

61

decrease in body weight. On the other hand, in all groups of C. sinensis

treatment as well as in reversibility group there is increase in body

weight.

In the present study also there is increase in the body weight of C.

sinensis co-treated rats but this increase is less than the increase reported

in the normal control group.

Yung His- Kao and co-workers (2000) have reported reduction in

the body weight with 2-7 days treatment with catechins of green tea. The

effect of epigallocatechin on body weight is dose dependant. Our studies

are not in agreement with these studies. The reason for difference might

be due to the difference in experimental design.

On the other hand Puming He (2001) reported no association

between the final body weights or body weight gain and the action of

green tea extract (30 gm/kg dose). Macdonald et al. (2010) have also

reported no significant changes in the body weight of experimental

animals.

During the present project liver was studied. The weight of liver

showed variation from the normal control group.

The weight of liver showed decrease with 5 mg dose of C. sinensis.

But this decrease in liver weight was not significant. No plausible

explanation for this fact can be given at this point. On the other hand, in

10 mg dose group of C. sinensis, weight of liver was within normal range,

both after 15 days and 30 days durations.

Young His- Kao (2000) observed decrease in the liver weight with

different catechins. Puming He (2001) had also studied protection of liver

62

injury against lipoposacharide stress with green tea extract but they have

not reported increase or decrease in the liver weight.

Discussion on the effects on liver biochemistry

In the liver, levels of total proteins, albumin, globulin, cholesterol,

ALT and AST were evaluated. During the present project no significant

change was observed in the level of total proteins and level of albumin

and globulin separately in the liver, after 15 and 30 days treatment in both

sets with 5 mg dose of C. sinensis, whereas with 10 mg dose of C.

sinensis after 15 and 30 days treatment in both sets, the level of total

proteins and level of albumin and globulin separately were almost in

normal range.

Maruthi et al. (2010) had reported increase in the protein and

albumin levels in the liver with CCl4 + Azima tetracantha extract. These

changes suggest the stabilization of endoplasmic reticulum leading to

protein synthesis.

Level of cholesterol showed decrease with 5 mg dose of C. sinensis

after 15 and 30 days treatment in both sets but this decrease was not

significant. In 10 mg dose group normal level of cholesterol was observed

in all treatment groups.

Arulmozhi et al. (2010) studied the protective effect of Solanum

nigrum fruit extract (SNFEt) against chronic ethanol toxicity in albino

rats. The level of total cholesterol was significantly increased in ethanol-

fed rats. Co-administration of SNFEt progressively improved the

cholesterol level towards normal.

63

As a major organ of the antioxidant defense system, the liver plays

a pivotal role in the regulation of lipoprotein transport and cholesterol

biosynthesis. Phospholipids are the vital components of biomembranes

and mainly act as regulators of membrane-bound enzymes important in

determining the pathology of alcoholism (Frayn, 1993).

Assessment of liver function can be made by estimating the

activities of ALT and AST, which are enzymes originally present in

higher concentration in cytoplasm. When there is any liver damage, the

level of these enzymes increases.

In the present project the level of alanine transaminase (ALT) and

aspartate transaminase (AST) showed no significant decrease with 5 mg

dose of C. sinensis in both sets for 15 days and 30 days in comparison to

alcohol control group. Whereas these parameters were almost in normal

range with 10 mg dose of C. sinensis in both sets for 15 days and 30 days

duration when compared with normal control groups of rats. It indicates

normal function and structure of liver, although we have not studied liver

histology.

High levels of the alanine transaminase (ALT) and aspartate

transaminase (AST) in the liver are also confirmatory of the liver damage.

Their high concentration indicates extent of liver damage (Sheil Sherlock

and Dooley, 1993). Dahiru et al. (2007) have used aqueous extract of

Zizipus mauritiana leaves along with alcohol and reported decreased

levels of ALT and AST.

Venukumar and Latha had also reported decline in the level of

ALT and AST (that was increased in CCl4 control groups) in

CCl4+Coscinium fenestratum treated rats thus indicating the

64

hepatoprotective effect of C. fenestratum. Liver histology of such

experimental animals also showed improvement. In their study these

biochemical tests were supported by histopathological observations of

liver sections.

These findings are in agreement with our findings, although

histological studies were not conducted during the present project.

Discussion on the effects on various blood parameters

During the present study the blood parameters showed marked

changes after treatment with 5 mg and 10 mg doses of aqueous extract of

C. sinensis co treated with 0.25 ml and 0.5 ml doses of ethanol. In

experimental animals treated with 5 mg dose of C. sinensis co-treated

with 0.25 ml and 0.5 ml doses of ethanol, the haemoglobin percentage,

RBCs count and PCV% showed non significant increase after 15 days

and significant increase after 30 days compared with alcohol treated

groups of rats and showed decrease in comparison of normal control

groups. But with 10 mg dose of C. sinensis the Hb%, RBCs count and

PCV% showed significant increase in comparison of alcohol treated rats

and almost normal when compared with normal control groups, whereas

the level of significance were different in all durations of both sets.

In the present study, we investigated the natural antioxidants

present in C. sinensis and evaluated for the first time the effect of C.

sinensis against ethanol-induced toxicity in rats. The obtained results

showed that C. sinensis has potential antioxidant activity.

Grinberg et al. (1997) had reported protection of red blood cells

against oxidative damage by tea polyphenols. Yung-His-Kao (2000)

studied the effects of different catechins of C. sinensis although the

65

experimental design is different from ours. They reported increase in

PCV, RBC, Hb % with ECG and EGCG. During the present project also,

all these levels showed increase, if we compare them with ethanol control

groups. The main difference is that during the present project C. sinensis

extract was given along with ethanol treatment and secondly we used

whole extract, not different fractions like Yung-His-Kao’s group. Our

results are consistent with findings of above workers.

El-kott et al. (2008) reported that administration of malathion

caused no significant decrease in Hb% and significant (p<0.05) decrease

in RBCs count when compared to control groups of same duration.

Administration of malathion + green tea extract caused increase in Hb%

and RBCs count when compared to malathion treated groups.

Mongi Saoudi et al. (2011) reported that Opuntia vulgaris fruit

extract (OE) treatment caused significant increase in the levels of RBC

and Hb% against alcohol induced toxicity when compared with

methanol-treated group. They suggested that the protective effects of OE

may be due to the modulation of antioxidant enzymes activities. Our

findings are also in agreement with these researchers.

In the present study we have also calculated MCV, MCH and

MCHC percentage and as these are based on the Hb%, RBC count and

PCV %, the levels of these parameters were also changed according to

these values.

The animals treated with 5 mg dose of C. sinensis co-treated with

0.25 ml and 0.5 ml doses of ethanol, the MCV, MCH and MCHC

percentage showed non significant increase after 15 days and significant

increase after 30 days compared with alcohol treated groups of rats and

66

showed decrease in comparison of normal control groups. But with 10 mg

dose of C. sinensis the MCV, MCH and MCHC percentage showed

significant increase in comparison of alcohol treated rats and almost

normal when compared with normal control groups. The levels of

significance were different in all durations of both sets.

The total leucocyte count (TLC) showed increase with 5 mg dose

of C. sinensis co-treated with 0.25 ml and 0.5 ml doses of ethanol after 15

days but this increase was non significant. After 30 days treatment the

level were almost normal with both 5 mg and 10 mg doses of C. sinensis

when compared with normal control groups in all durations, whereas the

level of significance was different in both sets.

DLC also showed changes according to the change in TLC but in 5

mg C. sinensis group number of neutrophils and eosinophils was

comparatively lower than the normal control group. Percentage of

monocytes and basophils was comparatively unaffected in all treated

groups. In 10 mg C. sinensis group of both sets, DLC was observed

normal after 15 days as well as 30 days.

Leucocyte count is reported to be decreased with both catechins

ECG and EGCG by Yung-His-Kao and coworkers (2000). During the

present project also we observed decrease in the number of leucocytes

with C. sinensis treatment in comparison to normal control. These results

appear quite similar and indicate towards same mode of action of C.

sinensis. The normal levels of RBCs, Hb % and leucocytes in 10 mg C.

sinensis co treated with 0.25 ml and 0.5 ml doses of ethanol, indicate that

no damage to RBCs or WBCs is caused by ethanol, when C. sinensis

extract is given along with ethanol. In presence of polyphenols and

67

catechins in C. sinensis aqueous extract probably ethanol is not able to

exert its toxic effects.

Level of blood sugar showed no significant decrease in 5 mg dose

of C. sinensis aqueous extract co-treated with both doses of ethanol in

both sets for different durations in comparison of alcohol treated groups

and remained increased when compared with normal control groups.

When we studied blood sugar in all groups of 10 mg C. sinensis

treatment, the level of blood sugar was almost in normal range in

different durations of both sets.

Our results are consistent with Mongi Saoudi et al. (2011) who

reported Opuntia vulgaris fruit extract (OE) treatment could significantly

decrease the level of glucose, when compared with methanol-treated

group. These results suggested that OE could exhibit a potential source of

natural antioxidants against methanol-induced biochemical disruption in

rats. The protective effects of OE may be due to the modulation of

antioxidant enzymes activities and inhibition of lipid per oxidation. Same

mode of action can be suggested for C. sinensis.

Level of proteins showed no significant increase with 5 mg dose of

C. sinensis co-treated with 0.25 ml and 0.5 ml doses of ethanol after 15

days and 30 days. Significant (p<0.001) increase was observed with 10

mg dose after 15 and 30 days in both sets in comparison of alcohol

treated rats and when we compared 10 mg dose of C. sinensis groups with

normal control groups of rats in all durations of both sets, the level of

proteins was normal.

Mehana and co-workers (2010) observed same decrease in the

level of total proteins, albumin and globulin with green tea extract in rats

68

co-treated with lead. As alcohol can disturb protein synthesis in

hepatocytes especially of albumin, it is natural that level of total proteins,

albumin and globulin will be reduced in blood of alcohol treated rats but

co-treatment with C. sinensis extract causes some improvement

indicating protective role of green tea against alcohol induced damage.

Mongi Saoudi et al. (2011) observed that Opuntia vulgaris fruit extract

(OE) treatment caused significant increase in the level of total proteins

when compared with methanol-treated group.

There is also evidence of protective role of green tea extract against

Chlorpyriphos induced liver toxicity in rats and improvement in the level

of serum albumin, globulin and total proteins (Heikel and co-workers,

2013). The increase in serum albumin, globulin and total proteins might

be either due to the production of enzymes lost as tissue damage or to

meet increase demand for detoxifying the alcohol.

During the present study urea level showed no change with either 5

mg or 10 mg dose of C. sinensis in comparison to alcohol treated and

normal control groups in both sets and all durations.

In present work level of cholesterol showed decrease with 5 mg

dose of C. sinensis co-treated with 0.25 ml and 0.5 ml doses of ethanol

after 15 days and 30 days but it was non significant. But significant

(p<0.001) decrease was observed with 10 mg dose after 15 days in both

sets in comparison to alcohol treated groups and when we compared

results of all groups of 10 mg dose of C. sinensis with normal control

groups of rats of both sets, the level of cholesterol was in normal range.

Level of bilirubin showed no significant decrease with 5 mg dose

of C. sinensis co-treated groups of all durations of both sets in

69

comparison of alcohol treated rats. On the other hand significant

(p<0.001) decrease was observed with 10 mg dose after 15 days in both

sets in comparison of alcohol treated rats. When we compare 30 days

treatment of 10 mg dose of C. sinensis co-treated with 0.25 ml and 0.5 ml

doses of ethanol, with normal control groups of rats, the level of bilirubin

was almost in normal range.

Dahiru and Obidoa (2007) studied the effect of the aqueous extract

of Ziziphus mauritiana leaves in chronic alcohol-induced liver damage.

They reported increase in hepatic bilirubin level in comparison to normal

control groups. Pre-treatment of rats with aqueous extract of Z.

mauritiana along with alcohol administration resulted in significant

(p<0.05) reduction of bilirubin level compared to the group exposed to

alcohol only. Our results are also consistent with Maruthi et al., 2010 and

Wan-Guo Yu et al. 2011.

When blood parameters of some enzymes such as acid

phosphatase, alkaline phosphatase, SGOT or serum ALT and SGPT or

serum AST were studied in experimental animals, fed on 5 mg and 10 mg

doses of C. sinensis for various durations, different picture was obtained

of different enzymes.

During the present study acid phosphatase level showed no

significant decrease with 5 mg dose of C. sinensis co-treated with 0.25 ml

and 0.5 ml doses of ethanol in all durations when compared with alcohol

treated rats. On the other hand, with 10 mg dose significant decrease was

observed after 15 days in both sets in comparison of alcohol treated rats

and when we compared 10 mg dose of C. sinensis groups after 30 days

treatment with normal control groups of rats of both sets, the level of acid

phosphatase was in normal range.

70

In tissues, acid phosphatase is associated with break down and

catalytic activities. High level of acid phosphatase is observed in tissues

with cell destruction and lytic activities. Normal level of acid phosphatase

in blood clearly indicates that no tissue damage is taking place.

It is relevant to note here again that levels of various enzymes in

blood are treated as a sensitive index of liver injury as well as injury to

other tissues, because enzymes released from cells eventually circulate in

the blood.

Level of ALP, ALT and AST showed decrease with 5 mg dose of

C. sinensis co-treated with 0.25 ml and 0.5 ml doses of ethanol after 15

days but it was non significant. After 30 days treatment, significant

decrease was observed in ALP and ALT in comparision with alcohol

treated rats. Level of all these three enzymes showed significant decrease

with 10 mg dose of C. sinensis co-treated with both doses of ethanol after

15 days and 30 days in comparison of alcohol treated rats but the level of

significance was different for various durations. When we compared

readings of 10 mg dose of C. sinensis groups with normal control groups

of rats for all durations of both sets, the level of these parameters were

almost in normal range. Rees and Sinha (1960) had extensively studied

serum glutamate pyruvate transaminase and serum glutamate

oxaloacetate transaminase during chronic liver toxicity.

ALP is a key enzyme involved in trans-phosphorylation and

membrane transport. Several pathophysiological factors have known to

affect alkaline phosphatase activity (Verma, 1980) and co-treatment with

C. sinensis was able to regulate alkaline phosphatase activity, hence

normal levels of alkaline phosphatase were observed in the blood.

71

Liposaccahride is thought to induce the apoptosis of liver cells,

through the action of TNF-α in GaIN-sensitized mice. It has been

demonstrated that apoptosis precedes the necrosis of liver cells and

increase in acid phosphatase, ALT and AST activities (Leist et al. 1995).

Puming, He et al. (2001) studied effects of green tea against

liposaccahride (LPS) induced liver injury and reported protecting effect

of green tea.

Dahiru and Obidoa (2007) who studied the effect of the aqueous

extract of Ziziphus mauritiana leaves in chronic alcohol-induced liver

damage and observed that pretreatment of rats with aqueous extract of

Ziziphus mauritiana with alcohol administration resulted in significant

(p<0.05) reduction of the elevated level of ALT and AST when compared

to the group exposed to alcohol only.

Mehana and co-workers (2010) while working with green tea

extract against lead induced toxicity reported improvement in the

elevated levels of ALP, ALT and AST. These enzymes were significantly

reduced in lead and green tea extract treated groups in comparison to lead

treated groups alone. Almost parallel results are obtained during the

present study.

Since aminotransferases (ALT and AST) are of important class of

enzymes linking carbohydrates and amino acid metabolism, the

relationship between the intermediate citric acid cycle is well established.

These enzymes are regarded as markers of liver injury. ALP is membrane

bound enzyme and alteration in its activity is likely to affect the

membrane permeability and produce derangement of the transport of the

metabolites. The elevated activity of ALP is related with adaptation of

liver to damaging factors.

72

Naik et al., (2011) and Shahid et al. (2012) reported that the

elevated serum marker enzymes (AST, ALT, and ALP) along with histo-

architectural changes in both CCl4 and ISO treated rats confirm the onset

of liver and cardiac necrosis.

Heikel and co-workers (2013) observed improvement in the levels

of ALP, ALT and AST when rats were co-treated with green tea and

Chlorpyriphos. The activities of ALP, ALT and AST enzymes are the

more sensitive biomarkers directly implicated in the extant of hepatic

damage and toxicity. The elevated levels of these enzymes in the ethanol

treated groups can be due to the release of these enzymes from the

cytoplasm into the blood circulation indicating necrosis and inflammatory

reaction. The polyphenols and catechins present in green tea are able to

prevent this damage thus causing decrease in the level of these marker

enzymes, in comparison to ethanol treatment alone.

It has been reported that co treatment with antioxidants protects

liver and other body organs hence lower level of these enzymes. Green

tea was able to protect damage of liver cells as is evident by decrease in

the ALT, AST, ALP levels.

Discussion on the Effects of Camellia sinensis

Aqueous Extract Co-treated with Ethanol and Its

Comparison with Normal Control and Ethanol

Control Groups

During the present study it was tried to investigate the protective

effects of Camellia sinensis against ethanol toxicity. C. sinensis has been

evaluated for its antioxidant, anticarcinogenic, antiangiogenic, antiviral

73

and bactericidal properties by a number of researchers (Chen, J., 1992,

Hertog et al, 1997, Imai, 1997, Wang et al., 1992, Kuroda and Hara,

1999, Cheng and Lin, 2002,). Much of the effects of green tea are

believed to be mediated by the polyphenolic constituents most notably

epigallocatechin-3-gallate (EGCG) present in it. (Katiyar and Mukhtar,

1996).

Herbs and medicinal plants have been used throughout the world

for centuries to treat many diseases and 80% of the world population

relies on botanical preparations as medicine for their health need. The

biological activity of a natural product is very often believed to be the

result of the combined action of several of its constituents.

Fruits, vegetables, common beverages, grains, many marine

products, medicinal plants and herbs posses diversified pharmacological

properties and contain nutraceuticals with the potential to protect against

various diseases.

During the present study we have tried to investigate the protective

effects of C. sinensis against the alcohol toxicity. It is already a well

established fact and we also possess data that alcohol causes damage to

cells and tissues of the body and we have already discussed these effects

and compared with the work off other research groups.

Discussion on the effects on body weight and organ weight

During the present study results showed decrease in the body

weight in ethanol-fed rats with 0.25 ml and 0.5 ml doses in all durations

when compared to healthy rats. It is obvious that chronic ethanol

administration produced toxicity in rats, which was evident by weight

loss.

74

Gruchow et al 1985 and Lieber, 2003 have reported no significant

change in body weights of ethanol treated rats when compared with the

controls. Venukumar and Latha (2004) have reported loss in body weight

in carbon tetra chloride treated rats. Aruna et al., 2005 and Das et al.,

2006 reported significantly decrease body weight due to alcohol when

compared to control rats. Nagaraja et al. (2006) also reported decrease in

the body weight of alcohol treated rats although this gain is statistically

insignificant. However, Geidam (2007) have reported slight increase in

the body weight. Dahiru and Obidoa (2007) observed significant decrease

in body weight of rats treated with alcohol for four weeks. Arulmozhi et

al. (2010) also found that the body weight was significantly reduced in

the ethanol-treated rats. Mac Donald et al. (2010) and Osonuga et al.

(2010) have also reported decrease in mean body weight of rats treated

with ethanol and that this decrease was non significant. Pratt and

Thomson (1992) reported that excessive alcohol intake can impair the

utilization of nutrients by altering their storage and excretion. The relative

decrease in mean body-weight recorded in ethanol treated rats may be

adduced to malnutrition resulting from reduced absorption of nutrients

from the intestine. Leiber C.S. (2000) also reported that excessive alcohol

ingestion disturbs the metabolism of most nutrients in the diet resulting in

primary or secondary malnutrition. Malnutrition may be caused by either

maldigestion or malabsorption and impaired utilization of nutrients,

which leads to significant weight loss.

In contrast when ethanol-fed rats were given aqueous extract of

green tea, the body weight was restored in a dose dependant manner to

near normal level with 10 mg dose of C.sinensis in all treatment groups.

Ethanol treatment alone causes decrease in body weight so it appears that

green tea exerts improvement in body weight.

75

Venukumar and Latha (2004) have studied protective effect of

Coscinium fenestratum against hepatotoxicity caused by CCl4 and

reported loss in body weight in carbon tetra chloride treated rats as

compared to normal controls. They have also reported weight gain in

animals treated with C. fenestratum extracts and CCl4.

Dahiru and Obidoa (2007) studied the protective effects of aqueous

extract of Ziziphus mauritiana leaves against alcohol induced liver

damage and observed significant decrease in body weight of rats treated

with alcohol compared to normal group at the end of 4 weeks. The rats

pre-treated with 400mg/kg body weight aqueous extract of Z. mauritiana

gained body weight when compared with group ingested with alcohol

only. This indicates that pre-treatment with Z. mauritiana extract

decreased weight loss due to chronic alcohol ingestion.

Arulmozhi et al. (2010) studied the antioxidant and

antihyperlipidemic effect of Solanum nigrum fruit extract (SNFEt)

against chronic ethanol toxicity and found that body weight was

significantly reduced in the ethanol-treated rats as compared to the

controls. Supplementation of SNFEt (250 mg/kg b.wt) along with ethanol

increased the body weight to near normal, which suggests the protective

effect of SNFEt against ethanol toxicity.

But it is evident that drinking green tea causes loss of body weight,

as reported by Yung-his-kao et al. (2000) and Puming He et al. (2001)

and it is considered as one of the beneficial properties of green tea.

During the present study weight of liver showed some increase in

ethanol fed rats with 0.25 ml and 0.5 ml doses in all durations when

compared to normal control group of rats.

76

The increase weight of liver is attributed to the fact that chronic

alcohol consumption causes accumulation of lipids and proteins in

hepatocytes. The liver can be injured by many chemicals and drugs. In

the present study ethanol was selected as a hepatotoxicant to induce liver

damage, since it is clinically relevant. Ethanol produces a constellation of

dose-related deleterious effects in the liver (Leo et al., 1982). In chronic

alcoholics, hepatomegaly occurs due to accumulation of lipids and

proteins in the hepatocytes, (Ashakumary et. al., 1993) with an impaired

protein secretion by hepatocytes (Vilstrup et al., 1985). There was

significant increase in liver weight due to damage caused by

administration of CCl4 in control animals as compared to normal group

.Water is retained in the cytoplasm of hepatocytes leading to enlargement

of liver cells resulting in increased total liver mass and volume (Blancho

et al., 1990).

Gujarati et al. (2006) studied the hepatoprotective effects of

alcoholic and aqueous extract of leaves of Tylophora indica (Linn.) in

rats. They reported increase in the weight of liver of rats treated with 3.76

g/kg ethanol for 25 days. This alcohol induced increase in total liver

weight was prevented by treatment with Tylophora indica leaf extract,

thus indicating a hepatoprotective effect.

Nagaraja et al.(2006) reported increase in the weight of liver of rats

treated with ethanol as compared to the controls. Javed et al. (2008)

observed increase in the liver weight of quails with 2% ethanol but

decrease in the liver weight after 4 weeks. After statistical analysis they

have concluded no significant difference from control groups. Naik et al.

(2011) reported that carbon tetrachloride treatment significantly increased

77

the relative weight of liver of rats compared to control rats, which may be

due to hepatomegaly or hepatotrophy.

Arulmozhi et al. (2010) found that the liver weight was increased

in animals fed with ethanol as compared to the controls. The liver weight

was found to be elevated in the ethanol-treated rats as compared to the

controls. When ethanol-fed rats were given Solanum nigrum fruit extract

(SNFEt), the liver weight was restored in a dose dependant manner to

near normal level during the experimental period.

Naik et al. (2011) studied protective effect of curcumin on CCl4

induced hepatotoxicity in rats. Carbon tetrachloride treatment

significantly increased the relative weight of liver of rats as compared to

the controls, which may be due to hepatomegaly or hepatotrophy.

Pretreatment with curcumin (200mg/kg,b.w.) in CCl4 induced liver injury

significantly prevented the increased relative weight of liver. The

protective effect of curcumin is further supported by a significant

reduction in CCl4-induced hepatomegaly/hypertrophy of liver in rats.

Arunmozhi et al. (2013) showed that administration of Chara

parpam has affected on liver weight in experimental rats with liver

damage induced by CCl4.

In contrast when ethanol-fed rats were given aqueous extract of

green tea, the liver weight was decreased in a dose dependant manner to

near normal level in 10 mg dose of C. sinensis in all treatment groups.

Ethanol treatment alone also causes increase in liver weight so it appears

that green tea exerts improvement in liver function.

Discussion on the effects on biochemical parameters of liver

78

By keeping in mind the beneficial effects of green tea

administration on liver damage, this study is designed to evaluate the

hepatoprotective effect of green tea against alcohol induced damage in

experimental rats.

Green tea, one of the most popular beverages of the world contains

polyphenolic antioxidants. The major organ involved in ethanol toxicity

is liver and different workers have reported that the severity of lesions

was also highest in liver.

Herbal medicines derived from plant products are increasingly

being used to treat various medical problems. Plant extracts have been

used by traditional medical practitioners for the treatment of liver

disorders for centuries. It is being acknowledged that plants contain wide

beneficial health effects.

The liver cirrhosis is the result of late stage scaring in chronic liver

disorder. This inflammation is produced by free radicals generated by

viruses, toxins, unhealthy fats, alcohol and some drugs that are attaking

liver cell (Tsukamoto et. al., 1997).

The green tea polyphenols prevent oxygen free radicals induced

hepatocyte lethality thus preventing the liver injury (Cai et. al., 2002).

The protective effects of green tea extracts or tea polyphenols against

liver fibrosis and liver cirrhosis in rats have been reported (Bun et. al.,

2006). An increased consumption of green tea may reduce the risk of

liver disease (Jin et. al., 2008).

We have studied total proteins, cholesterol, ALT and AST

parameters in the liver by preparing tissue homogenate.

79

During the present study results of protein level showed significant

decrease with 0.25 ml and 0.5 ml doses of ethanol treatment when

compared to normal control groups of rats in all durations.

Decreased level of serum protein and albumin is indicative of liver

damage. This damage is attributed to the fact that higher concentration of

alcohol dehydrogenase enzyme which catalyses alcohol to aldehyde is

found in the liver of alcoholics (Tussy and felder, 1989). Chronic alcohol

use results in the accumulation of export type proteins due to inhibition of

the secretion of the proteins from liver. It results in decreased levels of

protein (Baraona, 1982). Ethanol induced injury at least in part to an

oxidative stress resulting from the increase in free radical production and

/or decrease antioxidant defense. Dubey et al. (1994) reported that

capacity of liver to synthesize albumin is adversely affected by

hepatotoxins. They reported low level of albumin in alcoholic cirrhosis.

The lowered level of total proteins in the liver of ethanol treated rats can

be attributed to this fact only. It is also worthwhile to mention here that

level of albumin was highly significantly reduced in liver where as

globulin was not much affected. Inhibition of protein synthesis is an

important indication of cellular damage. Decreased level of proteins also

indicates that new cells are not being formed. Arun Raj et al. (2009) have

also observed decrease in the level of serum albumin. Lower level of

serum albumin may be due to reduced liver function.

Mehana et al. (2010) reported that the plasma levels of total protein

and albumin were significantly low in liver homogenate in lead treated

rats in comparison with control group.

During the present study in contrast when ethanol-fed rats were

given 5 mg and 10 mg doses of aqueous extract of green tea, the

80

decreased level of protein was restored in a dose dependant manner to

normal level.

Shalan et al. (2005) reported lead binds to plasmatic proteins where

it causes alterations in a high number of enzymes and perturb protein

synthesis in hepatocytes. Mehana et al. (2010) studied ameliorated

effects of green tea extract on lead induced liver toxicity in rats and

reported that the plasma levels of total proteins and albumin were

significantly low in liver homogenate in lead treated rats when compared

with control group. In lead and green tea group the plasma levels of total

proteins and albumin were significantly higher than lead treated rats. The

oral supplementation of green tea to lead intoxicated rats augmented the

antioxidant potential and reduced the tissue injury of liver cells. Maruthi

et al. (2010) have also reported increase in the protein and albumin levels

in liver with CCl4 + Azima tetracantha extract.

During the present study results of cholesterol level showed

significant increase with both doses of ethanol when compared to normal

control groups of rats in all durations. Five mg dose of C. sinensis caused

no significant decrease in 15 and 30 days, but significant decrease was

observed with 10 mg dose of C. sinensis and cholesterol levels were

normal.

Arulmozhi et al. (2010) also studied effect of Solanum nigrum fruit

extract (SNFEt) on the cholesterol level of control and ethanol-

administered rats. The level of total cholesterol was significantly

increased in ethanol-fed rats. Co-administration of SNFEt progressively

improved the cholesterol level towards normal when compared to control

group.

81

As a major organ of the antioxidant defense system, the liver plays

a pivotal role in the regulation of lipoprotein transport in plasma and

cholesterol biosynthesis. Hence, the alteration in the membrane

composition may be the reason for the toxic effects caused by ethanol.

Assessment of liver function can be made by estimating the

activities of ALT and AST, which are enzymes originally present in

higher concentration in cytoplasm. When there is any liver damage, the

level of these enzymes increases. High levels of the alanine transminase

(ALT) and asparate transaminase (AST) in the liver are also confirmatory

of the liver damage. Their high concentration indicates extent of liver

damage (Sheil Sherlock and Dooley, 1993).

In present project the level of alanine transminase (ALT) and

asparate transaminase (AST) showed non significant decrease with 5 mg

dose of C. sinensis in both sets for 15 days and 30 days. Whereas these

parameters were almost in normal range with 10 mg dose of C. sinensis in

both sets for 15 days and 30 days compared with normal control groups

of rats. It indicates normal function and structure of liver, although we

have not studied liver histology.

Dahiru et al. (2007) have used aqueous Zizipus mauritiana leaves

along with alcohol and reported decreased levels of ALT and AST.

Venukumar and Latha had reported decline in the level of ALT and

AST (that was increased in CCl4 control groups) in CCl4+Coscinium

fenestratum treated rats. Liver histology of such experimental animals

also showed improvement. These biochemical observations were

supported by histopathological experiment of liver sections.

82

These findings are in agreement with our findings, although

histological studies were not conducted during the present project.

Yang et al. (2010) studied the hepatoprotective effects of apple

polyphenols (AP, Appjfnol) against CCl4-induced acute liver damage in

Kunming mice. CCl4 caused increase in the levels of ALT and AST in the

hepatic homogenate. Apple polyphenols significantly prevented the

increase in serum ALT and AST levels in acute liver injury induced by

CCl4 and produced a marked amelioration in the histopathological hepatic

lesions, which may be due to its free radical scavenging effect, inhibition

of lipid peroxidation, and its ability to increase antioxidant activity.

Mehana et al. (2010) studied ameliorated effects of green tea

extract on lead induced liver toxicity in rats and reported that the liver

enzymes, ALT and AST activities were significantly elevated in lead

treated rats in comparison with controls. These enzymes were

significantly reduced in Pb + green tea extract-treated rats when

compared with Pb-treated rats.

The liver enzymes aminotransferases (ALT and AST) are an

important class of enzymes linking carbohydrate and amino acid

metabolism. These enzymes are regarded as markers of liver injury. In

addition, ALP is membrane bound and its alteration is likely to affect the

membrane permeability and produce derangement in the transport of

metabolites. Moreover, elevated ALP activity, which was used as a

marker of liver adaptation to damaging factors, has been reported

frequently in Pb-exposed animals (Gill et al., 1991; Moussa and

Bashandy, 2008). Shivashankara et al. (2012) observed that dietary

agents prevent the alcohol induced hepatotoxicity. Dietary agents

like Allium sativum (garlic), Camellia sinensis (tea), Glycine max

83

(soyabean) etc. protect against ethanol-induced hepatotoxicity and shown

that the beneficial effects of these phytochemicals in preventing

the ethanol induced hepatotoxicity are mediated by the antioxidant

effects. Parallel results have been obtained during the present work with

green tea extract against alcohol toxicity.

Discussion on the effects on various blood parameters

Since erythrocytes circulating in the peripheral blood share in part

the structural and functional features common to most cells, it was

thought worthy to study effects of the ethanol on blood cells as well as

the protective effects of Camellia sinensis. While maintenance of the

normal erythrocytic constituents e.g. enzymes, haemoglobin, glutathione

and cations is crucial for the internal functions, prevention of any damage

to the cells depends upon membrane integration of erythrocytes. It is this

very membrane which obviously is a major target for interaction with

toxic factors. In principle, toxic haematological manifestations are the

outcome of two major causes –

1. The agent exerts a direct, primary, destructive action on the

peripheral red cells, as a result of which the viability of the cells is

affected.

2. The damaging effects on erythrocytes are secondary, resulting from

a primary action of the toxicant on the erythropoietic (blood

forming) organs.

The destructive or suppressive effect on erythrogenic tissue can be

expressed as a failure in red cell production and in accelerated destruction

of peripheral erythrocytes in the presence of an abnormal cell population.

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Decrease in Hb%, RBC count and PCV % were observed during

the present work with ethanol treatment for 15 and 30 days as compared

to normal control animals. In contrast when ethanol-fed rats were co-

treated with both 5 mg and 10 mg doses of aqueous extract of green tea,

the decreased level of Hb%, RBC count and PCV % were restored in a

dose dependant manner to normal level.

Grinberg et al. (1997) have reported protection of red blood cells

against oxidative damage by tea polyphenols. Yung-His-Kao (2000)

studied the effects of different catechins of C. sinensis although the

experimental design is different from ours. They reported increase in

PCV, RBC, Hb % with ECG and EGCG. During the present project also,

all these levels showed increase, if we compare these with ethanol control

groups, when we compare these readings with that of normal control

groups, the normal levels of all these parameters were observed in C.

sinensis and alcohol co-treated groups. The main difference is that during

the present project C. sinensis extract was given along with ethanol

treatment and secondly we used whole extract, not different fractions like

Yung-His-Kao’s group, because in daily life, whole green tea extract is

consumed. Our results are consistent with the findings of above workers.

El-kott et al. (2008) reported that administration of malathion

caused non significant decrease in Hb% and significant (p<0.05) decrease

in RBCs count when compared to control groups. Administration of

malathion + green tea extract also caused increase in Hb% and RBCs

count when compared to treated groups. Our findings are in agreement

with these authors.

Mongi Saoudi et al. (2011) observed that Opuntia vulgaris fruit

extract (OE) treatment caused significant increase in the levels of RBCs

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and Hb% against alcohol induced toxicity when compared with

methanol-treated group. They suggested that the protective effects of OE

may be due to the modulation of antioxidant enzymes activities.

Hichem et al. (2012) studied protective effect of Opuntia ficus

indica f. inermis (prickly pear) juice upon ethanol-induced damages in rat

erythrocytes. Ethanol administration also reduced the scavenging activity

in plasma and enhanced erythrocytes hemolysis, as compared to control

rats. In animals also given prickly pear juice during the same

experimental period, the studied parameters were much less shifted. This

protective effect was found to be dose-dependent. It is likely that the

beneficial effect of the extract is due to the high content of antioxidant

compounds.

The MCV, MCH and MCHC percentage are based on the Hb%,

RBC count and PCV%. The level of these parameters was also changed

according to these values.

The animals treated with 5 mg dose of C. sinensis co-treated with

both doses of ethanol i.e. 0.25 ml and 0.5 ml, the MCV, MCH and

MCHC percentage showed some increase after 15 days and significant

increase after 30 days when compared with alcohol treated groups of rats

i.e. these readings were approaching normal level. But with 10 mg dose

of C. sinensis the MCV, MCH and MCHC percentage showed significant

increase in comparison of alcohol treated rats and were almost normal

when compared with normal control groups.

The number of total leucocyte count (TLC) showed decrease with

alcohol treatment but when experimental animals were co-treated with 5

mg dose of C. sinensis and 0.25 ml and 0.5 ml doses of ethanol,

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respectively after 15 days no significant increase was observed and after

30 days treatment significant increase was reported in comparison of

alcohol treated rats and almost normal with the treatment 10 mg dose of

C. sinensis caused the level of TLC towards normal, when compared with

normal control groups in all durations.

DLC also showed changes according to the change in TLC but in

5 mg C. sinensis group number of neutrophils and eosinophils was

comparatively lower than the normal control group. Percentage of

monocytes and basophils remained unaffected in all treated groups. In 10

mg C. sinensis group of both sets, DLC was observed normal after 15

days as well as 30 days.

Leucocyte count is reported to be decreased with both catechins

ECG and EGCG by Yung-His-Kao and coworkers (2000). During the

present project we did not observed decrease in the number of leucocytes

with C. sinensis treatment. The normal levels of RBCs, Hb % and

leucocytes in 10 mg C. sinensis co treated with 0.25 ml and 0.5 ml

ethanol respectively indicate that no damage to RBCs or WBCs is caused

by ethanol, when C. sinensis extract is given along with ethanol. In the

presence of polyphenols and catechins in C. sinensis probably ethanol is

not able to exert its toxic effects.

This effect appears to be dose dependent and duration dependent.

Less increase (towards normalcy) was observed after 15 days treatment

with 0.25 ml +5 mg dose and more increase was observed after 30 days

treatment with 0.25 ml +10 mg dose of C. sinensis.

In reversibility study also almost same pattern was observed. In 15

days and 30 days reversibility group of set I, normal levels of TLC and

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DLC were observed. In reversibility studies of set II also, normal TLC

and DLC were observed.

Eichner and Hillman (1971) observed that ethanol depresses the

hemopoiesis in an organism by producing vacuolization in the

granulocyte precursors of the bone marrow. Neutropenia has been

reported in patients consuming alcohol. Sipp et al. (1993) has reported

that eosinopenia after ethanol treatment may be due to the direct effect of

ethanol on adrenal gland secretion. Ethanol-induced corticosterone

secretion might be the cause of decreased eosinophil count. Nagaraja et

al.(2006) observed a significant decrease (p<0.001) in absolute neutrophil

and eosinophil counts after chronic ethanol administration for a treatment

period of 7 and 14 days in unstressed rats when compared to control

groups. Administration of ethanol with a dosage of 2g/kg body weight

resulted in more significant decrease in absolute neutrophil (p<0.05) and

absolute eosinophil counts (p<0.01 -7 days; p<0.05 -14 days). Our

findings are also in accordance with the findings of these researchers.

We have used whole extract of C. sinensis. The reason for using

whole extract was that green tea is commonly consumed in this form

only. According to our findings, ethanol treatment causes decrease in

Hb% and C. sinensis whole extract is able to bring back this decreased

Hb% to normal level. Ethanol treatment decreased number of RBCs and

treatment with whole extract of C. sinensis was able to increase RBC

number back to normal level. Same pattern is observed with haematocrit

volume or PCV. Alcohol treatment caused decrease in haematocrit

volume and treatment with aqueous extract of C. sinensis was able to

increase the value of haematocrit volume back to normal.

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Level of blood sugar showed significant increase with both doses

of ethanol as compared to normal control groups. There was some

decrease in 5 mg C. sinensis dose co-treated with both doses of ethanol in

both sets for different durations in comparison of alcohol treated rats and

some increase when compared with normal control groups. When we

studied blood sugar in all groups of 10 mg C. sinensis treatment, the level

of blood sugar was almost in normal range in different durations of both

sets. These findings indicate that antioxidant or flavanols and polyphenols

(catechins) present in C. sinensis probably inhibit ethanol uptake by

tissues and exert their protective effects.

Arun Raj et al. (2009) studied the biochemical effects of feeding

soft drink and ethanol. Rats were fed with ethanol and observed

significantly higher level of glucose (p<0.05) in comparison to control

rats.

Our results are consistent with Mongi Saoudi et al. (2011) who

reported that Opuntia vulgaris fruit extract (OE) treatment could

significantly decrease the level of glucose, when compared with increased

glucose level in methanol-treated group. These results suggested that OE

could exhibit a potential source of natural antioxidants against methanol-

induced biochemical disruption in rats. The protective effects of OE may

be due to the modulation of antioxidant enzyme activities and inhibition

of lipid per oxidation.

When we compare the values in reversibility groups in set I and set

II, the normal levels of sugar remained so even after discontinuation of

the C. sinensis extract feeding up to 15 days.

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During the present study serum protein and albumin levels were

decreased significantly (p<0.05) due to both doses of ethanol as

compared to normal control group of rats but globulin level remained

unaffected.

Baraona E. and Lieber C.S., (1986) reported that ethanol inhibits

the secretion of protein from liver and ultimately in blood. Chronic

ethanol abuse results in intrahepatic accumulation of export-type proteins

which results in decreased plasma levels. These effects appear to be

mediated by acetaldehyde. Das S.K. and Vasudevan D.M., (2005)

reported decreased serum protein and albumin levels and postulated that

liver damage starts after 30 days of ethanol exposure. Common feature of

chronic alcoholic liver disease is progressive hypoalbuminemia. Gujarati

et al. (2006), Geidam et al. (2007), Arun Raj et al. (2009) and Das et al.

(2009) observed significantly decreased total protein and albumin levels

due to ethanol exposure in comparison to rats of control group. Our

results are in agreement with the findings of above all authors.

K Al-Kubaisy and M Al-Noaemi (2007) studied the effects of the

essential oil of Nigella sativa for its antihepatotoxic effects in rats against

carbon tetrachloride (CCl4) toxicity. The decreased levels of proteins and

albumin observed in rats after treatment with CCl4 were found to be

increased in rats treated with N. sativa oil and CCl4 as compared to CCl4

alone treated rats. Maruthi et al. (2010), also reported the reduced levels

of total protein and albumin in CCl4 induced hepatotoxicity. The total

protein and albumin levels were raised with the Azima tetracantha leaves

extract suggesting the stabilization of endoplasmic reticulum leading to

protein synthesis.

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As alcohol can disturb protein synthesis in hepatocytes especially

of albumin, it is natural that level of total proteins, albumin and globulin

will be reduced in blood of alcohol treated rats but co-treated with C.

sinensis extract causes some improvement indicating protective role of

green tea against alcohol induced damage. Mongi Saoudi et al. (2011)

studied the methanol-induced haematological and biochemical toxicity in

rats. Methanol significantly decreased the levels of serum total protein.

They studied Opuntia vulgaris fruit extract (OE) treatment with

methanol, which caused significant increase in the level of total proteins

when compared with methanol-treated group.

There is evidence of protective role of green tea extract against

Chlorpyriphos induced liver toxicity in rats and improvement in the level

of serum albumin, globulin and total proteins (Heikel and co-workers,

2013). The increase in serum albumin, globulin and total proteins might

be either due to the production of enzymes lost as tissue damage or to

meet increase demand for detoxifying the alcohol. Our results are in

agreement with the findings of above all authors.

Level of serum cholesterol showed increase with all doses and

durations of ethanol treatment. When aqueous extract of C. sinensis was

introduced along with ethanol then level of cholesterol started decreasing.

Five mg dose reduced level of cholesterol to some extent after 15 days

and 30 days treatment but 10 mg dose of C. sinensis was able to reduce

elevated levels of serum cholesterol to normal levels. In 0.5 ml ethanol

group, level of serum cholesterol was more significantly elevated and 10

mg dose of C. sinensis was able to bring back elevated level of

cholesterol to normal level. In the reversibility group, when extract

feeding was discontinued for 15 days, the reduced level of serum

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cholesterol remained normal in all durations of 10 mg dose groups.

Ramirez and Gimenez (2002) also reported increase in circulating

cholesterol in serum in relation to control serum in ethanol treated rats.

Yung-Hsi-Kao and co workers (2000) reported reduction in serum

cholesterol with catechin EGCG. During the present study ethanol

treatment caused elevation in serum cholesterol level but this level was

decreased after treatment with different doses of C. sinensis aqueous

extract.

Atta et al. (2010) studied the hepatoprotective effect of methanol

extract of Zingiber officinale and Cichorium intybus against CCl4 induced

liver damage. Carbon tetrachloride treatment significantly elevated the

serum cholesterol concentration as compared to control group. Methanol

extract of ginger (250 and 500 mg/kg) and chicory (250 and 500 mg/kg)

given alone or mixed (1:1 wt/wt) restored the carbon tetrachloride-

induced alterations in the biochemical parameters. The hepatoprotective

effect of ginger and chicory was also confirmed by the histopathological

examination of liver tissue.

During the present study the level of urea showed no change after

15 and 30 days treatment with both doses of alcohol and with 5 mg and

10 mg doses of C. sinensis in comparison to alcohol treated and normal

control groups in both sets and all durations. Our results are consistent

with Das et al. (2009), also observed no changes in blood urea level after

treatment with ethanol till 4 weeks and present work duration also was for

30 days.

The level of bilirubin showed significant increase (p<0.01) after 15

and 30 days treatment with 0.25 ml and 0.5 ml doses in both sets

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compared to normal control groups. Level of bilirubin showed no

significant decrease with 5 mg dose of C. sinensis co-treated with ethanol

in all durations of both sets in comparison of alcohol treated rats. But

significant (p<0.001) decrease was observed with 10 mg dose after 15

days in both sets in comparison of alcohol treated rats and when we

compared the effect of 10 mg dose of C. sinensis groups co-treated with

both 0.25 ml and 0.5 ml doses of ethanol after 30 days with normal

control groups of rats, the level of bilirubin was almost in normal range.

Gujrati et al. (2006) and Dahiru and Obidoa (2007) also observed

that ethanol administration to rats resulted in significant elevation of total

serum bilirubin compared to control group. Our results are also consistent

with that of Maruthi et al., 2010 and Wan-Guo Yu et al. 2011.

Dahiru and Obidoa (2007) studied the effect of the aqueous extract

of Ziziphus mauritiana leaf in chronic alcohol-induced liver damage.

They reported increase in hepatic bilirubin level in comparison to normal

control. Pretreatment of rats with aqueous extract of Z. mauritiana with

alcohol administration resulted in significant (p<0.05) reduction of

bilirubin level when compared to the group exposed to alcohol only.

Wan-Guo Yu et al. (2011) demonstrated that 2′,4′-Dihydroxy-6′-

methoxy-3′,5′-dimethylchalcone has potential hepatoprotective effects

against CCl4 and decreased activity of total bilirubin. Which cause

attenuation of oxidative stress and inhibition of lipid peroxidation.

Acid phosphatase is a lysosomal enzyme which hydrolyses the

ester linkage in phosphate esters and help in the autolysis of the cell after

death. Experimental evidences show that it is not only restricted to

lysosomal fraction out has also been found in golgi cisternae and

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specialized region of endoplasmic reticulum. Acid phosphatase reaction

thus reflects some impressions about the structure and function of these

organelles or components.

During the present study level of acid phosphatase in blood showed

increase after 15 and 30 days treatment with 0.25 ml and 0.5 ml dose of

alcohol as compared to normal control group.

Ress and Sinha (1960) are of the opinion that the damaged organs

might produce an augmented quantity of the enzymes. Soliman et al.

(2006) studied that ethanol ingestion caused significant elevation in the

activity of enzyme acid phosphatase. Geidam et al. (2007) found mild

elevation in acid phosphatase enzyme in ethanol fed rats alone.

As activity of acid phosphatase is related with catalytic activities in

cell, effect of any toxicant or pollutant can increase level of acid

phosphatase. Similarly we observed increased level of acid phosphatase

with both doses and durations of ethanol.

During the present study acid phosphatase level showed some

decrease with 5 mg dose of C. sinensis co-treated with 0.25 ml and 0.5 ml

doses of ethanol in all durations when compared with alcohol treated rats.

But significant decrease was observed with 10 mg dose after 15 and 30

days treatment in both sets in comparison of alcohol treated rats and when

we compared these readings with normal control groups of rats of both

sets, the level of acid phosphatase was in normal range.

The level of enzymes ALP, ALT and AST was also increased

significantly in response to 15 and 30 days duration of ethanol exposure

with both dose groups as compared to normal control groups of the same

duration.

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Level of ALP, ALT and AST showed some decrease with 5 mg

dose of C. sinensis co-treated with both 0.25 ml and 0.5 ml doses of

ethanol after 15 and 30 days. The level of these enzymes showed

significant decrease with 10 mg dose of C. sinensis co-treated with both

doses of ethanol after 15 days and 30 days in comparison of alcohol

treated rats. When we compared the results of 10 mg dose of C. sinensis

with normal control groups of rats with same durations of both sets, the

level of these enzymes were almost in normal range.

ALP is a key enzyme involved in trans-phosphorylation and

membrane transport. Several pathophysiological factors have known to

affect alkaline phosphatase activity (Verma, 1980) and co-treatment with

C. sinensis was able to regulate alkaline phosphatase activity, hence

normal levels of alkaline phosphatase were observed in the blood.

Yung-His-Kao and co-workers (2000) studied effects of

epicatechin (ECG) and epigallocatechin (EGCG) of C. sinensis. They

reported no significant decrease in the level of SGPT (ALT) with

epicatechin (ECG) and epigallocatechin (EGCG) but they have reported

no significant increase in the level of SGOT (AST).

Puming, He. et al. (2001) studied effects of green tea against

liposaccahride (LPS) induced liver injury, liposaccahride is thought to

induce the apoptosis of liver cells, through the action of TNF-α in GaIN-

sensitized mice. It has been demonstrated that apoptosis precedes the

necrosis of liver cells and increase in acid phosphatase, ALT and AST

activities (Leist et. al. 1995).

Dahiru and Obidoa (2007) studied the effect of the aqueous extract

of leaves of Ziziphus mauritiana in chronic alcohol-induced liver damage

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and observed that pretreatment of rats with aqueous extract of Z.

mauritiana with alcohol administration resulted in significant (p<0.05)

reduction of elevated levels of ALT and AST in comparison to the groups

exposed to ethyl alcohol only.

Hepatotoxic action of CCl4 and leakage of liver enzymes into blood

have also been recorded by several investigators (Hikino et al., 1986;

Gadgoli and Mishra, 1995; 1999).

The effects of the essential oil Nigella sativa was studied for its

antihepatotoxic effects in rats against carbon tetrachloride (CCl4) which

induced hepatocytic damage. The increased levels of serum enzymes

(SGOT, SGPT and SAP) observed in rats treated with CCl4 were greatly

reduced in the animals treated with N. sativa oil and CCl4. These

biochemical observations were supported by histopathological test of

liver sections (K. Al-Kubaisy and M. Al-Noaemi, 2007). Mehana and co-

workers (2010) while working with green tea extract against lead induced

toxicity reported improvement in the elevated levels of ALP, ALT and

AST. These enzymes were significantly reduced in lead and green tea

extract treated groups in comparison to lead treated groups.

Since aminotransferases (ALT and AST) are important class of

enzymes linking carbohydrates and amino acid metabolism, these

enzymes are regarded as markers of liver injury. ALP is membrane bound

enzyme and alteration in its activity is likely to affect the membrane

permeability and produced derangement of the transport of the

metabolites. The elevated activity of ALP is related with adaptation of

liver to damaging factors.

96

Yang et al. (2010) observed apple polyphenols significantly

prevented the increase of serum ALT and AST levels in acute liver injury

induced by CCl4 and produced a marked amelioration in the

histopathological hepatic lesions.

Wan-Guo Yu et al. (2011) demonstrated that 2′,4′-Dihydroxy-6′-

methoxy-3′,5′-dimethylchalcone has potential hepatoprotective effects

against CCl4 toxicity and decreased activities of serum hepatic enzymes,

namely alanine aminotransferase, aspartate aminotransferase and alkaline

phosphatase, which cause attenuation of oxidative stress and inhibition of

lipid peroxidation. Naik et al. (2011) studied the effect of curcumin on

serum marker enzymes ALT, AST, and ALP in rats. The serum marker

enzymes were significantly elevated in both acute and sub-chronic CCl4-

induced liver injury as compared to control group of rats. Curcumin

treatment at different doses significantly restored the elevated level of

marker enzymes in different experimental groups.

Mehana et al. (2010) studied ameliorated effects of green tea

extract on lead induced liver toxicity in rats and reported that liver

enzymes, ALT, AST and ALP activities were significantly present in

blood elevated in lead treated rats in comparison with controls. These

enzymes were significantly reduced in Pb + green tea extract-treated rats

comparing with Pb-treated rats.

A.H. Atta et al. (2010) studied the hepatoprotective effect of

methanol extracts of Zingiber officinale and Cichorium intybus against

CCl4 toxicity. Carbon tetrachloride treatment significantly elevated the

ALT and AST activities compared to control group. Methanol extract of

ginger (250 and 500 mg/kg) and chicory (250 and 500 mg/kg) given

alone or mixed (1:1 wt/wt) restored the carbon tetrachloride-induced

97

alterations in these parameters. Shahid et al. (2012) reported that the

elevated serum marker enzymes (AST, ALT, and ALP) with CCl4 treated

rats confirm the onset of liver damage.

Heikel and co-workers (2013) observed improvement in the level

of ALP, ALT and AST when rats were co-treated with green tea and

chlorpyriphos.

All these findings are in agreement with the findings of the present

study. Normal levels of all these marker enzymes in tissues as well as in

blood indicate towards protection of cell and tissue injury caused by

ethanol. The protective effect of green tea extract appears to act in similar

manner as other antioxidants found in other plants such as soya

isoflavons and α-tocopherol.

The activities of ALP, ALT and AST enzymes are the more

sensitive biomarkers directly implicated in the extant of hepatic damage

and toxicity. The elevated levels of these enzymes in the ethanol treated

groups can be due to the release of these enzymes from the cytoplasm

into the blood circulation indicating necrosis and inflammatory reaction.

The polyphenols and catechins present in green tea are able to prevent

this damage thus causing decrease in the level of these marker enzymes,

in comparison to ethanol treatment alone.

This study revealed that ethanol ingestion perturbs the antioxidant

system and caused deleterious effects on animals.